Tadao Ishida

Aberrant DNA methylation associated with silencing BNIP3 gene expression in haematopoietic tumours

Hypoxia is a key factor contributing to the progression of human neoplasias and to the development of resistance to chemotherapy. BNIP3 is a proapoptotic member of the Bcl-2 protein family involved in hypoxia-induced cell death. We evaluated the expression and methylation status of BNIP3 gene to better understand the role of epigenetic alteration of its expression in haematopoietic tumours. Methylation of the region around the BNIP3 transcription start site was detected in four acute lymphocytic leukaemia, one multiple myeloma and one Burkitt lymphoma cell lines, and was closely associated with silencing the gene. That expression of BNIP3 was restored by treatment with 5-aza2′-deoxycytidine (5-aza-dC), a methyltransferase inhibitor, which confirmed the gene to be epigenetically inactivated by methylation. Notably, re-expression of BNIP3 using 5-aza2-dC also restored hypoxia-mediated cell death in methylated cell lines. Acetylation of histone H3 in the 5′ region of the gene, which was assessed using chromatin immunoprecipitation assays, correlated directly with gene expression and inversely with DNA methylation. Among primary tumours, methylation of BNIP3 was detected in five of 34 (15%) acute lymphocytic leukaemias, six of 35 (17%) acute myelogenous leukaemias and three of 14 (21%) multiple myelomas. These results suggest that aberrant DNA methylation of the 5′ CpG island and histone deacetylation play key roles in silencing BNIP3 expression in haematopoietic tumours.

Monoclonal antibodies as effective therapeutic agents for solid tumors

Monoclonal antibodies (mAbs) against growth factors or their receptors have been revealed to be effective therapeutic agents for solid tumors. Trastuzumab (humanized anti‐HER2 mAb) is the first mAb approved for the treatment of a solid tumor, metastatic breast cancer. Large‐scale phase III clinical trials are now ongoing to further evaluate the additive effects on chemotherapy and the efficacy as a maintenance monotherapy. Another anti‐HER2 mAb CH401 that we developed also seems to have good potential. This chimeric mAb completely suppressed the growth of established human tumor xenografts in SCID mice after a single injection. Furthermore, CH401 characteristically showed much stronger induction of apoptosis in HER2‐overexpressing gastric cancer cells compared to trastuzumab. Additional targets now being intensively evaluated are epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF). Both cetuximab (chimeric anti‐EGFR mAb) and bevacizumab (humanized anti‐VEGF mAb) have recently been shown to be of clinical value for metastatic colorectal cancer. Anti‐idiotype mAbs are unique as active immunotherapeutic agents, and survival benefits have been observed in clinical trials for solid tumors.

Genomic Screening for Genes Silenced by DNA Methylation Revealed an Association between RASD1 Inactivation and Dexamethasone Resistance in Multiple Myeloma

Purpose: Epigenetic changes such as DNA methylation play a key role in the development and progression of multiple myeloma. Our aim in the present study was to use genomic screening to identify genes targeted for epigenetic inactivation in multiple myeloma and assess their role in the development of resistance to dexamethasone. Experimental Design: Gene expression was examined using microarray screening, reverse transcription-PCR, and real-time quantitative PCR. DNA methylation was examined using bisulfite PCR, bisulfite sequencing, and bisulfite pyrosequencing in 14 multiple myeloma cell lines, 87 multiple myeloma specimens, and 12 control bone marrow samples. WST-8 assays were used to assess cell viability after treatment with 5-aza-2′-deoxycytidine and/or dexamethasone. Results: Microarray analysis was done to screen for genes up-regulated by 5-aza-2′-deoxycytidine. In RPMI8226 cells, 128 genes were up-regulated, whereas 83 genes were up-regulated in KMS12PE cells. Methylation of 22 genes with CpG islands in their 5′ regions, including RASD1, was confirmed. Methylation of RASD1 was associated with its inactivation, which correlated with resistance to dexamethasone. Treating multiple myeloma cells with 5-aza-2′-deoxycytidine restored sensitivity to dexamethasone. Methylation of RASD1 was also detected in a subset of primary multiple myeloma specimens, and the levels of methylation were increased after repeated antitumor treatments. Gene signature analysis revealed various genes to be synergistically induced by treatment with a combination of 5-aza-2′-deoxycytidine plus dexamethasone. Conclusion: Our findings indicate that epigenetic inactivation of genes, including RASD1, plays a key role in the development of dexamethasone resistance in multiple myeloma. Moreover, they show the utility of demethylation therapy in cases of advanced multiple myeloma.

Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells

By presenting immunogenic peptides at the cell surface, major histocompatibility complex (MHC) class II molecules play a key role in the control of adaptive immune responses. Whether expressed constitutively or induced by interferon-γ, expression of MHC class II molecules is regulated via coactivator class II transactivator (CIITA); moreover, suppression of their expression is one mechanism by which cancer cells escape host immunity. In this study, we surveyed the relationship between the expression of one MHC class II antigen, HLA–DR, and its coactivators in a group of haematopoietic cell lines, and explored the role of the aberrant DNA methylation in silencing HLA-DR expression. Among 26 cell lines studied, HLA-DR expression was lost from eight T-cell and two myeloid leukaemia cell lines, and this loss was closely associated with suppression of CIITA-PIV expression. Notably, nine of the 10 cell lines that lost CIITA-PIV expression showed methylation of the genes 5′ CpG island. Thus, DNA methylation is believed to inhibit the expression of MHC class II molecules in haematopoietic tumour cells by silencing its coactivator, CIITA-PIV. Furthermore, methylation of CIITA-PIV was detected in seven of 32 primary acute myeloid leukaemia specimens, indicating that epigenetic alteration is not a cell line-specific phenomenon. Collectively, these data suggest that, by suppressing expression of MHC class II molecules, epigenetic inactivation of CIITA provides a survival advantage to a subset of haematopoietic tumours.

A new Ehlers-Danlos syndrome with craniofacial characteristics, multiple congenital contractures, progressive joint and skin laxity, and multisystem fragility-related manifestations.

We previously described two unrelated patients showing characteristic facial and skeletal features, overlapping with the kyphoscoliosis type Ehlers–Danlos syndrome (EDS) but without lysyl hydroxylase deficiency [Kosho et al. (2005) Am J Med Genet Part A 138A:282–287]. After observations of them over time and encounter with four additional unrelated patients, we have concluded that they represent a new clinically recognizable type of EDS with distinct craniofacial characteristics, multiple congenital contractures, progressive joint and skin laxity, and multisystem fragility‐related manifestations. The patients exhibited strikingly similar features according to their age: craniofacial, large fontanelle, hypertelorism, short and downslanting palpebral fissures, blue sclerae, short nose with hypoplastic columella, low‐set and rotated ears, high palate, long philtrum, thin vermilion of the upper lip, small mouth, and micro‐retrognathia in infancy; slender and asymmetric face with protruding jaw from adolescence; skeletal, congenital contractures of fingers, wrists, and hips, and talipes equinovarus with anomalous insertions of flexor muscles; progressive joint laxity with recurrent dislocations; slender and/or cylindrical fingers and progressive talipes valgus and cavum or planus, with diaphyseal narrowing of phalanges, metacarpals, and metatarsals; pectus deformities; scoliosis or kyphoscoliosis with decreased physiological curvatures of thoracic spines and tall vertebrae; cutaneous, progressive hyperextensibility, bruisability, and fragility with atrophic scars; fine palmar creases in childhood to acrogeria‐like prominent wrinkles in adulthood, recurrent subcutaneous infections with fistula formation; cardiovascular, cardiac valve abnormalities, recurrent large subcutaneous hematomas from childhood; gastrointestinal, constipation, diverticula perforation; respiratory, (hemo)pneumothorax; and ophthalmological, strabismus, glaucoma, refractive errors.

MUC1 Mucin Core Protein Binds to the Domain 1 of ICAM-1

Background: MUC1 is aberrantly expressed on a variety of epithelial tumors. We have reported that MUC1 plays important roles in separation from primary site, invasion into the stromal tissue, and protection from immune responses. The aim of this study is to determine the precise binding of MUC1 to intercellular adhesion molecule 1 (ICAM-1) that accelerates the cancer metastasis. Methods: A cell aggregation assay between MUC1 cDNA transfectants and ICAM-1 expressing cells was employed. An anti-MUC1 antibody, anti-ICAM-1 antibody or synthetic peptide of MUC1 core protein was added to the assay to inhibit the cell aggregation. Results: MUC1 transfectants showed a significantly higher aggregation rate compared to the control cells. This aggregation was further enhanced by the inhibition of O-glycan biosynthesis. It was inhibited by either an anti-MUC1 antibody recognizing the tandem repeat domain of MUC1 core protein or an anti-ICAM-1 antibody identifying domain 1. It was also inhibited by a synthetic MUC1 peptide of 40 amino acids corresponding to two tandem repeats. Conclusions: The results revealed that a tandem repeat domain of MUC1 mucin core protein binds to domain 1 of ICAM-1, suggesting a potential role of MUC1- ICAM-1 interaction in the metastasis of epithelial tumors.

The analysis of interleukin-6 in patients with systemic IgG4-related plasmacytic syndrome—expansion of SIPS to the territory of Castleman's disease

vasculitis [8] and autoimmune thyroid disease [5]. High levels of lymph node mRNA expression and serum levels of TNFand IL-6 have been reported [9, 10], suggesting a hypercytokinaemia commonly seen in CTDs. Frequently, considerable effort must be spent in order to differentiate this entity from adult onset Still’s disease, RA and SLE [3, 5, 6]. The clinical outcome of AILT remains poor (median survival <3 years, 5-year survival 30–35%) [5]. Combination chemotherapy may lead to complete remission (50%) but relapse rates remain high. Overall combination chemotherapy appears to be superior to glucocorticosteroids alone [5] but studies are limited. Furthermore, some patients develop secondary diffuse large B-cell lymphoma (DLBCL) [3]. Recognition of AILT is therefore important as the management of the differential diagnoses may lead to inappropriate treatment. In summary, we present a rare case of AILT with clinical features of autoimmune disease. Rapid escalation and deescalation of the lymphadenopathy, combined with more prominent symptoms from the joints, gave the strong impression of an underlying disorder like RA. AILT should be considered in the differential diagnosis of late-onset RA or Still’s disease in older patients presenting with a recent history of lymphadenopathy and fever. In such patients, sites of lymph-node involvement nonapparent by physical examination should be investigated.

Incidence and Risk of Postherpetic Neuralgia after Varicella Zoster Virus Infection in Hematopoietic Cell Transplantation Recipients: Hokkaido Hematology Study Group

To assess the incidence of and risk factors associated with postherpetic neuralgia (PHN) after hematopoietic cell transplantation (HCT) varicella zoster virus (VZV) infection, we conducted a retrospective chart review of 418 consecutive patients who underwent HCT between April 2005 and March 2007. The male/female ratio was 221/197, median age at HCT was 47 years (range: 0-69 years), and autologous/allogeneic/syngeneic HCT ratio was 154/263/1. Seventy-eight patients developed VZV infection after HCT. Sixty-two patients had localized zoster, 11 patients had disseminated zoster (rash like chicken pox), and 4 patients had visceral zoster. All cases were treated with acyclovir (ACV) or valacyclovir (VACV), and there was no VZV infection-related death. Twenty-seven (35%) of the 78 patients with VZV infection suffered PHN after resolution of VZV infection. Multivariate analysis showed that advanced age is the only risk factor in autologous HCT (P = .0075; odds ratio [OR] = 1.14; 95% confidence interval [CI], 0.97-1.33). On the other hand, advanced age (P = .0097; OR = 1.06; 95% CI, 1.01-1.12), male gender (P = .0055; OR = 12.7; 95% CI, 1.61-100.1), and graft-versus-host disease (GVHD) prophylaxis with a tacrolimus-based regimen (P = .0092; OR = 9.56; 95% CI, 1.44-63.3) were associated with increased risk of PHN in allogeneic HCT. This study for the first time clarified the risk of PHN in HCT recipients.

Significance of erb B‐2 Gene Product as a Target Molecule for Cancer Therapy

The new monoclonal antibodies (MoAbs) E401, E811, E907 and E919 were prepared and characterized. These recognized an extracellular domain (amino acids No. 292–370) on the human c‐erbB‐2 gene product. Utilizing MoAb E811 and MoAb E919, a double determinant immunoassay (DDIA) was established to detect the soluble and the shed forms of the c‐erbB‐2 molecule. The levels of circulating erbB‐2 antigen in the sera of patients with benign diseases and healthy controls were very low. The incidence of positivity for shed c‐erbB‐2 antigen in gastric cancer, eolonic cancer, gall‐bladder caneer, pancreatic eancer and other cancers were 7.4%, 4.2%, 0%, 6.7% and 0%, respectively. Four of 54 patients with gastric carcinoma showed high levels of serum c‐erbB‐2 antigen. They belonged to clinical stage IV and their histological types were all well differentiated adenoeareinomas (two papillary and two tubular adenoeareinomas). Furthermore, the incidence of positive staining in gastric cancer was 34.6%; higher than that for shedding erbB‐2 antigen. Most of the cases v‐hich showed erbB‐2 expression on cells were well‐differentiated adenoeareinomas. Meanwhile, the distribution of erbB‐2 antigen was limited in normal tissues. The results suggest that the expression of erbB‐2 antigen is largely restricted to adenocarcinoma cells. It may not shed easily from these cells, and therefore It may be a very useful target molecule for passive immunotherapy.

Low-dose thalidomide plus low-dose dexamethasone therapy in patients with refractory multiple myeloma

We report the results of a non‐randomized phase II study of low‐dose thalidomide plus low‐dose dexamethasone therapy in 66 patients with refractory multiple myeloma. The overall response rate (near complete, partial and minimal response) was 63.6%, and progression‐free and overall survival periods were 6.2 and 25.4 months. In adverse events, the incidence of peripheral neuropathy and deep vein thrombosis was lower than the data reported in USA and Europe. On the other hand, leukopenia was observed in 41% of patients, including 11% of those with Grade 3. Leukopenia was closely related to pretreatment pancytopenia, especially thrombocytopenia. The incidence of adverse events related to dexamethasone was low. In conclusion, low‐dose thalidomide plus low‐dose dexamethasone therapy was as effective as high‐dose thalidomide plus high‐dose dexamethasone therapy in patients with refractory multiple myeloma. Leukopenia is one of the most serious adverse events in Japanese patients, especially in patients with pretreatment pancytopenia.